Genotyping of Mycobacterium leprae for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil.

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1-4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation.

Intrapatient comparison of Mycobacterium leprae by VNTR analysis in nasal secretions and skin biopsy in a Brazilian leprosy endemic region.

Background: This study compares the strains of genotypes of M. leprae from nasal secretions (NS) and skin biopsy (SB) in the same patient, supplementing conventional epidemiology to gain insight into the infection of leprosy in Fortaleza, Brazil. Methods: The sample consisted of 38 newly diagnosed leprosy patients attending the National Reference Center of Dermatology Dona Libania (CDERM), in Fortaleza, who tested positive for M. leprae by PCR in DNA extracts of nasal secretions. DNA was also extracted from skin biopsy (SB) scrapings of each patient and used for multiplex PCR amplification of M. leprae VNTR loci. The number of repeats at 15 loci were determined by the fragment length analysis method. Results: Locus VNTR genotypes were achieved in 38 NS, and in 38 SB specimens. M. leprae strains differed in their genotypes in paired specimens in all but two of 38 patients. The genotype similarity in the remainder ranged from 53% to 87%. Conclusion: M. leprae 15 VNTR loci genotypes of paired nasal and biopsy skin samples from five patients were identical, while as many as seven loci differed in the 33 other patients. When the NS and biopsy genotypes were pooled and compared, it was found that there was a great variability among different VNTR markers. It is important to investigate other molecular markers suitable for typing genetic variations of the bacilli.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil.

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Serum metabolomics reveals higher levels of polyunsaturated fatty acids in lepromatous leprosy: potential markers for susceptibility and pathogenesis.

BACKGROUND: Leprosy is a disease of the skin and peripheral nervous system caused by the obligate intracellular bacterium Mycobacterium leprae. The clinical presentations of leprosy are spectral, with the severity of disease determined by the balance between the cellular and humoral immune response of the host. The exact mechanisms that facilitate disease susceptibility, onset and progression to certain clinical phenotypes are presently unclear. Various studies have examined lipid metabolism in leprosy, but there has been limited work using whole metabolite profiles to distinguish the clinical forms of leprosy. METHODOLOGY AND PRINCIPAL FINDINGS: In this study we adopted a metabolomics approach using high mass accuracy ultrahigh pressure liquid chromatography mass spectrometry (UPLC-MS) to investigate the circulatory biomarkers in newly diagnosed untreated leprosy patients. Sera from patients having bacterial indices (BI) below 1 or above 4 were selected, subjected to UPLC-MS, and then analyzed for biomarkers which distinguish the polar presentations of leprosy. We found significant increases in the abundance of certain polyunsaturated fatty acids (PUFAs) and phospholipids in the high-BI patients, when contrasted with the levels in the low-BI patients. In particular, the median values of arachidonic acid (2-fold increase), eicosapentaenoic acid (2.6-fold increase) and docosahexaenoic acid (1.6-fold increase) were found to be greater in the high-BI patients. SIGNIFICANCE: Eicosapentaenoic acid and docosahexaenoic acid are known to exert anti-inflammatory properties, while arachidonic acid has been reported to have both pro- and anti-inflammatory activities. The observed increase in the levels of several lipids in high-BI patients may provide novel clues regarding the biological pathways involved in the immunomodulation of leprosy. Furthermore, these results may lead to the discovery of biomarkers that can be used to investigate susceptibility to infection, facilitate early diagnosis and monitor the progression of disease.

Genetic diversity of mycobacterium leprae isolates from Brazilian leprosy patients.

INTRODUCTION: Leprosy is a chronic disease caused by infection with Mycobacterium leprae, an obligate intracellular parasite. A problem in studying the transmission of leprosy is the small amount of variation in bacterial genomic DNA. The discovery of variable number of tandem repeats (VNTRs) allowed the detection of strain variation in areas with a high prevalence of leprosy. Four genotypes of M. leprae based on three single-nucleotide polymorphism (SNPs) were also discovered to be useful for analysis of the global spread of leprosy. METHODS: In this present study, we examined the allelic diversity of M. leprae at 16 select VNTR and three SNP loci using 89 clinical isolates obtained from patients mainly from the neighbouring states of São Paulo and Rio de Janeiro Brazil. RESULTS AND CONCLUSION: By use of a PCR-RFLP-based procedure that allows the recognition of SNP types 3 and 4 without the need for the more expensive DNA sequencing steps, characterisation of the main M. leprae genotypes was easy. When applied on the study population, it was found that the SNP type 3 is most frequent in these two states of Brazil, and that VNTRs provided further discrimination of the isolates. Two Short Tandem Repeats (STRs) were monomorphic, with the remaining 14 STRs represented by two to 18 alleles. Epidemiological associations with township or state were not evident in this random collection and require further investigations. In phylogenetic trees, branches formed by all 16 STRs clearly separated SNP type 3 organisms from the other types while the allelic patterns of two minisatellite loci 27-5 and 12-5 were highly correlated with SNP type 3. This strain typing study provide the basis for comparison of M. leprae strain types within Brazil and with those from other countries, and informed selection of genomic markers and methods for future studies.

[Study of rifampin and dapsone resistance in three patients with recurring leprosy]. / Estudio de resistencia a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra.

OBJECTIVE: To detect the presence of rifampin- and dapsone-resistant strains of Mycobacterium leprae in three patients with recurring leprosy and clinically-suspected antimicrobial resistance through molecular techniques. METHODS: A retrospective, descriptive study was conducted of three multibacillary patients at the "Agua de Dios" Sanitarium in Cundinamarca, Colombia, that presented leprosy relapses that were documented by medical history, bacilloscopy, and biopsy. Biopsies were taken of the skin lesions and the bacteria were subject to DNA extraction and purification. Regions of the rpoB and folP1 genes associated with antimicrobial resistance were amplified and subjected to touch-down polymerase chain reaction and the amplified products were sequenced using the Sanger method. RESULTS: A punctual mutation was identified in nucleotide 1367 of the rpoB gene in two of the samples studied. This mutation was not found in the folP1 gene of any of the three patients. CONCLUSIONS: The mutation identified showed strains of rifampin-resistant M. leprae in two of the three patients with recurring leprosy. Mutations that indicate dapsone-resistance were not detected in any of the three patients.

Estudio de resistencia a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra / Study of rifampin and dapsone resistance in three patients with recurring leprosy

OBJETIVO: Detectar la presencia de cepas de Mycobacterium leprae resistentes a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra y sospecha clínica de resistencia antimicrobiana, mediante la aplicación de técnicas moleculares. MÉTODOS: Se realizó un estudio descriptivo retrospectivo en tres pacientes multibacilares del Sanatorio de Agua de Dios, Cundinamarca, Colombia, que habían presentado recidivas de lepra documentadas por su historia clínica, baciloscopia y biopsia. Se obtuvieron biopsias de lesiones cutáneas que se procesaron para la extracción y purificación del ADN bacilar. Se amplificaron regiones de los genes rpoB y folP1 asociadas con la resistencia antimicrobiana, mediante la reacción en cadena de la polimerasa "touch-down" y se secuenciaron los productos amplificados mediante el método de Sanger. RESULTADOS: Se detectó una mutación puntual en el nucleótido 1367 del gen rpoB en dos de las muestras estudiadas. No se encontró la mutación estudiada en el gen folP1 en ninguno de los tres pacientes. CONCLUSIONES: La mutación identificada demostró la presencia de bacilos de M. leprae resistentes a la rifampicina en dos de los tres pacientes estudiados con recurrencia de la enfermedad. No se detectó la mutación indicadora de resistencia a la dapsona en ninguno de los tres pacientes.

OBJECTIVE: To detect the presence of rifampin- and dapsone-resistant strains of Mycobacterium leprae in three patients with recurring leprosy and clinically-suspected antimicrobial resistance through molecular techniques. METHODS: A retrospective, descriptive study was conducted of three multibacillary patients at the "Agua de Dios" Sanitarium in Cundinamarca, Colombia, that presented leprosy relapses that were documented by medical history, bacilloscopy, and biopsy. Biopsies were taken of the skin lesions and the bacteria were subject to DNA extraction and purification. Regions of the rpoB and folP1 genes associated with antimicrobial resistance were amplified and subjected to touch-down polymerase chain reaction and the amplified products were sequenced using the Sanger method. RESULTS: A punctual mutation was identified in nucleotide 1367 of the rpoB gene in two of the samples studied. This mutation was not found in the folP1 gene of any of the three patients. CONCLUSIONS: The mutation identified showed strains of rifampin-resistant M. leprae in two of the three patients with recurring leprosy. Mutations that indicate dapsone-resistance were not detected in any of the three patients.

Identification and distribution of Mycobacterium leprae genotypes in a region of high leprosy prevalence in China: a 3-year molecular epidemiological study.

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been proposed as a means of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population are lacking. To this end, a study was initiated to assess the diversity and distribution of prevalent Mycobacterium leprae strains in Qiubei County, Yunnan Province, People's Republic of China, where the annual detection rate of leprosy is 10-fold higher than the national average rate. Sixty-eight newly diagnosed leprosy patients were included in the study. MLVA at eight M. leprae loci was applied using DNA extracts from skin biopsies. The number of alleles per locus ranged from 4 to 24, providing adequate strain discrimination. MLVA strain typing identified several clusters of patients whose M. leprae specimens shared similar VNTR profiles. Two of these clusters were comprised of patients who resided predominantly in the north and northwest parts of Qiubei County. Furthermore, it was found that multicase families are common in this county: 23 of the 68 patients were from 11 families. Intrafamilial VNTR profiles closely matched within six families, although they were different between the families. Moreover, VNTR patterns related to those found in some multicase families were also detected in patients in the same or adjacent townships, indicating the utility of VNTR strain typing to identify and detect short-range transmission events. Social contact through village markets is proposed as a means of transmission.

Comparative analysis of B- and T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture filtrate protein 10.

Culture filtrate protein 10 (CFP-10) from Mycobacterium tuberculosis is a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells from M. tuberculosis-infected mice and humans. The sequence of the Mycobacterium leprae homologue of CFP-10 shows only 40% identity (60% homology) at the protein level with M. tuberculosis CFP-10 and thus has the potential for development as a T- or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinant M. leprae and M. tuberculosis CFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for either M. leprae or M. tuberculosis CFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against either M. leprae or M. tuberculosis CFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections of M. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting that M. leprae CFP-10, like its homologue in M. tuberculosis, is a secreted protein.

Multiple polymorphic loci for molecular typing of strains of Mycobacterium leprae.

The need for molecular tools for the differentiation of isolates of Mycobacterium leprae, the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of M. leprae have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of M. leprae propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of M. leprae and enhances the potential of this technique to track the transmission of leprosy.

Characterization of the epitope of anti-lipoarabinomannan antibodies as the terminal hexaarabinofuranosyl motif of mycobacterial arabinans.

mAb CS-35 is representative of a large group of antibodies with similar binding specificities that were generated against the Mycobacterium leprae lipopolysaccharide, lipoarabinomannan (LAM), and which cross-reacted extensively with LAMs from Mycobacterium tuberculosis and other mycobacteria. That this antibody also cross-reacts with the arabinogalactan (AG) of the mycobacterial cell wall, suggesting that it recognizes a common arabinofuranosyl (Araf)-containing sequence in AG and LAM, is demonstrated. The antibody reacted more avidly with 'AraLAM' (LAM with naked Araf termini) compared to 'ManLAM' (in which many Araf termini are capped with mannose residues) and mycolylarabinogalactan-peptidoglycan complex (in which the terminal Araf units are substituted with mycolic acids). Neither did the antibody bind to AG from emb knock-out mutants deficient in the branched hexa-Araf termini of AG. These results indicate that the terminal Araf residues of mycobacterial arabinan are essential for binding. Competitive ELISA using synthetic oligosaccharides showed that the branched hexa-Araf methyl glycoside [beta-D-Araf-(1-->2)-alpha-D-Araf-(1-)(2)-(3 and 5)-alpha-D-Araf-(1-->5)-alpha-D-Araf-OCH(3)] was the best competitor among those tested. The related linear methyl glycoside, beta-D-Araf-(1-->2)-alpha-D-Araf-(1-->5)-alpha-D-Araf-(1-->5)-alpha-D-Araf-OCH(3), representing one linear segment of the branched hexa-Araf, was less effective and the other linear tetrasaccharide, beta-D-Araf-(1-->2)-alpha-D-Araf-(1-->3)-alpha-D-Araf-(1-->5)-alpha-D-Araf-OCH(3), was ineffective. The combined results suggest that the minimal epitope recognized by antibody CS-35 encompasses the beta-D-Araf-(1-->2)-alpha-D-Araf-(1-->5)-alpha-D-Araf-(1-->5)-alpha-D-Araf within the branched hexa-Araf motif of mycobacterial arabinans, whether present in LAM or AG.